Vaccination against host cell-associated herpes viruses

ABSTRACT

The invention relates to the field of so-called “host cell-associated herpes viruses,” such as Marek&#39;s disease-like virus (MDV) of poultry and Varicella Zoster virus (VZV) of man, and to vaccination against disease caused by these viruses. The invention provides a vaccine directed against an infection caused by a herpes virus that is essentially host cell-associated comprising a recombinant viral genome derived from the herpes virus, the genome allowing recombination essentially free of the host cell.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of PCT International PatentApplication No. PCT/EP/01/08893, filed on Aug. 1, 2001, designating theUnited States of America, and published, in English, as PCTInternational Publication No. WO 02/12288 A3 on Feb. 14, 2002, thecontents of the entirety of which is incorporated herein by thisreference.

TECHNICAL FIELD

The invention relates to the field of vaccination against so-called hostcell-associated herpes viruses such as Marek's disease virus (MDV) ofpoultry and Varicella Zoster Virus (VZV, causing chickenpox and zosterafter reactivation from latency) of man and to vaccination againstdisease caused by these viruses, and, in particular, relates to poultrydisease, in particular to the field of vaccination against Marek'sdisease.

BACKGROUND

In particular, Marek's disease has been a problem of the poultryindustry from the beginning of intensive production of poultry meat. Itis a herpes viral disease that is causing a large variety of clinicalsigns starting from immunosuppression, neurological disorders, anemiaand unspecified apathies and ending with severe lymphatic cancers atlater stages of infection. In the beginning of the history of Marek'sdisease, there were no treatments and no preventive measures. Then anapathogenic-related (Serotype 3) virus was isolated from turkeys (HVT)and was initially used for vaccination.

However, some time after introduction of vaccination with HVT, Marek'sdisease emerged again and it became clear that the circulating fieldviruses had changed to circumvent the protection induced by the HVTstrain. At this time, a new apathogenic virus was discovered (Rispensstrain), which in general has the same serotype as the viruses causingdisease. This vaccine strain was introduced very rapidly into the marketand produced very good vaccination results.

However, again, after about ten years, new outbreaks of diseaseoccurred; again, circulating field viruses had changed to circumvent theprotection induced by the vaccine strain in current use. Then acombination of both vaccines (HVT and Rispens) was used to protect theanimals; however, satisfactory results were only seen temporarily.Currently, new outbreaks of disease occur despite all thesevaccinations. The reason for this is not yet understood but there is aclear need for the introduction of new potent vaccines.

Problems associated with vaccinations against Marek's disease are that,despite the fact that Marek vaccines have been produced for a longperiod, the method of preparation of the vaccines could not be improved.The reason for this is that, in general, the essentially hostcell-associated virus can be grown essentially only in primary hostcells such as in the case of MDV or HVT in primary cells such asfibroblasts prepared from poultry, such as chickens, free of pathogensand, in the case of Varicella Zoster Virus, in (essentially primary)human cells (again, of course, free of contaminating pathogens) andcannot or only with great difficulties be achieved out of context of thespecific cell of the respective host. This makes, in general, a vaccinedirected against viral infections or disease caused by these types ofviruses difficult, if not nearly impossible on a practical level, toproduce and thus expensive.

For example, the Rispens vaccine directed against Marek's disease, whichis at present considered the only sufficiently potent one, is, as allserotype-1 Marek viruses, strictly host-cell associated. Infectivity ofthe cell-associated virus (such as, for example, serotype 1 and 2) iscompletely lost during normal freezing or lyophilization. Therefore, thepreparation of the vaccine includes a very complicated and expensivestep, where whole cells must be frozen in liquid nitrogen. The vaccinemust be stored and transported and kept under liquid nitrogen until useand therefore causes tremendous costs and problems during transport.

Then, at the site of use, the vaccine must be used very carefully, sincethe infected cells are very sensitive to environmental factors. Factorssuch as elevated temperatures, exposure to light and residual detergentsin glassware used often damage the virus such that no sufficientlyviable vaccine batch can be prepared, leading to complete vaccinefailures. Such failures can be recognized only when the disease alreadystarts to break out and the affected poultry show symptoms of disease.

In short, up to now, all attempts to provide inactivated, subunit orrecombinant vaccines to protect against Marek's disease failed and,therefore, there is currently no alternative to live, cell-associatedvaccines comprising Marek's Disease Virus. Marek's disease remains to becontrolled by application of infected-cell preparations as a vaccine.These preparations not only contain living cells suspended inDMSO-containing media and the whole variety of cellular antigens, butthey also have to be stored in liquid nitrogen. Consequently, the coldchain has to be maintained from vaccine production to the vaccine userand until administration. In addition, once thawed, the vaccine has tobe administered within a very short period of time and every bird has tobe injected. Several of these problems are shared by those wishing toprepare vaccines against other essentially cell-associated herpesviruses such as Varicella Zoster Virus.

Marek's disease virus (MDV) is a member of the Alphaherpesvirinaesubfamily of the Herpesviridae (Lee et al., 2000; Murphy et al., 1995).Based on virulence for the chicken and ability to induce T celllymphomas, MDV is generally grouped into three serotypes (MDV-1, MDV-2,and MDV-3). MDV-3 represents the herpes virus of turkeys (HVT) which waswidely used for vaccination against MDV-related disease. However, aftervaccination failures and development of so-called virulent or veryvirulent MDV-1 (Witter, 1985), attenuated MDV-2 strains and laterattenuated MDV-1 strains (e.g., strain CVI 988 Rispens) were used invaccine formulations (Witter, 1985). In recent years and first reportedin the United States, even more virulent MDV-1, so-called very virulentplus (vv+), and MDV-1 variants appeared and caused high incidence ofMarek's disease and mortality caused by tumor development andimmunosuppression early after infection (Witter, 1997). One vv+ strain,584A, was passaged more than 100 times on chicken embryo fibroblasts(CEF) and was shown to loose pathogenicity for chickens (Witter, 1997).However, the molecular basis for the increased pathogenicity of vv+MDV-1 and similarly for loss of virulence are poorly understood becausemolecular analyses of MDV-1 are difficult to perform. On the one hand,no or only small amounts of infectious virus progeny is released incultured cells; on the other hand, production of MDV-1 recombinants islaborious and, due to the highly cell-associated nature of the agent incell culture, multiple rounds of purification of virus recombinants areneeded (Cantello et al., 1991; Sakaguchi et al., 1993; Parcells et al.,1994; Schat et al., 1998; Anderson et al., 1998).

On top of that, as mentioned already above, vaccination cannot guaranteeto protect the animals from all Marek's disease field viruses. Thevirus—as all Herpes viruses—is capable of finding ways to escape theimmune response induced by the vaccines. Therefore, rapid adaptation ofvaccines to the field situation would be needed. Currently, this is doneby isolation of field isolates (such as HVT or Rispens) and/or furtherattenuation in vitro. The isolation itself is causing tremendousproblems because of the difficulties of getting the cell-associatedinfectious virus out of chickens and infecting cells in cell culture.The attenuation steps that would follow are very laborious and timeconsuming, especially since plaque purification is extremely difficult,which is again due to the cell-associated nature of the virus.

The result from attenuation is normally not defined. As a result ofthese facts, no vaccines that would provide relief where current HVT-and Rispens-type vaccines fail have entered the market for a long time.In addition, often an over-attenuation occurs during vaccine productionsince the virus has been passaged for too many times. This furtheraggravates the low efficacy of the HVT- and Rispens-type vaccines in thefield. In short, the following problems constitute a large part of thecurrent impasse in MDV control. There is a low reproducibility ofclassical vaccine production; one sees over-attenuation of vaccinevirus, undefined attenuation of vaccine virus, high production costs,high storage and transport costs, high sensitivity of the vaccine toenvironmental factors, and a too slow development of new vaccine strainsespecially for cell associated viruses.

These problems are compounded by the fact that now circulating fieldviruses give rise to high antibody titers in poultry production stock,whereby these high antibody titers are given through the progeny viamaternal antibody in the eggs. The influence of these maternalantibodies during initial infection by current vaccine virus furtherdecreases the current efficacy of vaccination against Marek's disease.

DISCLOSURE OF THE INVENTION

The invention provides a vaccine directed against an infection caused bya herpes virus that is essentially host-cell associated comprising arecombinant viral genome derived from the herpes virus, the genomeallowing recombination essentially free of the host cell. To thateffect, the invention herewith provides a recombinant viral genomederived from a herpes virus that is considered to be essentially hostcell associated, the genome preferably capable of at least some measureof replication in the host cell and at the same time allowingrecombination essentially free or independently of the host cell.Homologous recombination in eukaryotic cells is no longer required. Inthe detailed description, such a genome is provided for a Marek'sdisease-like virus.

Therein, as an example, a genome of Marek's disease virus serotype 1(MDV-1), strain 584Ap80C, was cloned in Escherichia coli as a bacterialartificial chromosome (BAC). BAC vector sequences were introduced intothe Us2 locus of the MDV-1 genome by homologous recombination afterco-transfection of chicken embryo fibroblasts (CEF) with viral DNA andrecombinant plasmid pDS-pHA1 which contained BAC sequences and theEco-gpt gene instead of the MDV-1 Us2 gene and flanking sequences.Transfection progeny was passaged on CEF cells in the presence ofmycophenolic acid and xanthine/hypoxanthine. After four rounds ofselection, viral DNA was prepared and used to transform Escherichia colistrain DH10B. Several colonies harboring the complete MDV-1 genome wereidentified. These MDV-1 BACs were transfected into CEF cells and from 3days after transfection, infectious MDV-1 was recovered. Growth of MDV-1recovered from various BACs was indistinguishable of that of theparental virus as assayed by plaque formation and determination ofgrowth curves.

The invention thus provides a method for producing or obtaining arecombinant essentially host-cell associated herpes viral genomecomprising (if required near complete or complete) infectious herpesviral nucleic acid derived from an MDV and/or VZV isolate.

Now that the essentially complete genome is obtained free of the hostcells, it was originally thought to be firmly associated. The inventionalso provides a genome that allows full application of all recombinanttechniques available to the person skilled in the art of molecularbiology and, for example, thus also provides a vaccine at leastcomprising a (replicative) minigenome.

For example, the invention provides a minigenome that provides for theexpression of only a couple of glycoproteins (such as gB, gC, gD orcombinations thereof) and, for example, ICP4 or another gene productthat has been shown to induce cellular immunity in herpes viruses. Sucha minigenome serves to identify genes that are important in protection,considering that replication of the genome in (eukaryotic) host cells isno longer provided. Adding, for example, a HCMV or SV40 promoter infront of each gene or gene construct would provide for the finalidentification of a minimal protective unit. For a replication-competentminigenome, the invention also provides deleting the entire US region,or a major part thereof, whereby the resulting minivirus replicates alsoin host cells.

In another embodiment, the invention provides such a genome whichcomprises an essentially full-length copy derived from the herpes virus,“essentially full-length” herein indicating that a great part of thegenes of the viral genome is present, except for some that arepreferably (at least functionally) left out such as a gene essential forreplication or spread of the virus in a host or host cell culture, asprovided herein in the detailed description. For example, in one of therecovered genomes according to the invention, BAC20, sequences encodingglycoprotein B (gB) were deleted by one-step rece-mediated mutagenesisusing a linear DNA fragment. Glycoprotein B-negative MDV-1 reconstitutedafter transfection of gB-negative BAC20 DNA (20DgB) were only able togrow on cells providing gB in trans, demonstrating that gB is essentialfor MDV-1 growth in cultured host cells. Other genes essential forgrowth and for which cells can be provided which produce the geneproduct in trans are gH, ICP4, UL15, UL28 and UL9, or other genesconsidered essential for growth such as those listed below.

Furthermore, the invention provides use of a genome for the preparationof a vaccine. In one embodiment, such a vaccine is directed against adisease caused by an infection with an essentially host cell-associatedherpes virus; however, in another embodiment, such a vaccine may be usedas a vector vaccine and may comprise other or additional pathogens ornucleic acid stretches encoding therefor. For MDV, preferred additionalpathogen nucleic acid comprises nucleic acid derived from, for example,Newcastle Disease virus, Eimeria spp, Salmonella spp, chicken infectiousanemia virus, influenza virus, infectious bursal disease virus,reovirus, or other pathogens commonly seen in poultry.

Thus, the invention also provides a vaccine wherein the genome comprisesa functional deletion in a gene essential for replication and/or spreadof the herpes virus in a host cell, or wherein the viral genome at leastcomprises a nucleic acid encoding an antigenic substance capable ofeliciting an (preferably essentially protective) immune response againstan infection of an individual with the herpes virus. A typical essentialgene or fragment thereof to be deleted can, for example, be an MDVhomologue of UL1=glycoprotein L; UL5; UL8; UL9; UL15; UL18; UL19;UL22=glycoprotein H; UL26; UL26.5; UL27=glycoprotein B; UL28; UL29;UL30; UL52; UL53; ICP4 or genes or fragments thereof selected from theUS region of the genome (FIG. 1).

In a preferred embodiment, the invention provides a vaccine comprising afunctional deletion in a gene essential for eliciting a marker immuneresponse specific for the herpes virus allowing immunologicaldiscrimination between an individual vaccinated with the vaccine and anindividual infected with the essentially cell associated herpes virus.Preferred marker responses are, for example, directed at gC, gM, gD, orgE, whereby the detailed description further explains (here in the caseof gM) such a deletion in a gene essential for eliciting a marker immuneresponse.

Furthermore, the invention provides a vaccine wherein the viral genomeat least comprises a nucleic acid encoding a proteinaceous substancecapable of modulating transcription and/or translation of a nucleic acidencoding an antigenic substance capable of eliciting an immune responseagainst an infection of an individual with the herpes virus.

Preferably, the vaccine comprises an essentially full-length copyderived from the herpes virus to maintain as many functions required foreffective modulation of transcription and/or translation of the vaccinegenome in the vaccinated host; however, minigenomic vaccination is alsoprovided herein. It is, of course, preferred to efficiently modulatetranscription and/or translation of a nucleic acid encoding a foreignpathogen, or antigenic substance derived thereof, when expressing anadditional pathogen or an antigenic substance derived thereof from thegenome, and it may be contemplated that also foreign pathogens (i.e.,non-herpes virus regulatory elements) are provided to the genome whenproviding a vaccine according to the invention provided with a nucleicacid encoding an additional pathogen.

In particular, the invention provides a vaccine wherein the herpes viruscomprises Marek's disease-like virus. In particular, is it preferred toprovide a vaccine wherein the Marek's disease-like virus comprisesserotype 1. Also, now that methods for manipulation of the genomeinvolved beyond the context of the host cell with which the genomeoriginally was associated are provided, a vaccine is provided that,instead of being derived from normal attenuated or a-virulent isolatesof Marek's disease-like virus, is derived instead from a virulent, avery virulent or a very virulent plus field-virus, because rapidisolation of infectious clones from field isolates is now possible,allowing preparation of DNA vaccines for prevention of Marek's diseasein chicken and turkeys, where mutations into the genome can beintroduced very rapidly. The same system can be used also for otheressentially cell-associated Herpes viruses like Varicella Zoster Virus.

The use of replicative viral genomes as provided herein containing partsof or entire and infectious Marek's Disease virus (MDV-1) genomes opensa variety of new possibilities to generate more efficacious,biologically safe and stable MDV-1 vaccines. Owing to the fact thatrecombinant MDV-1 are recovered from cloned DNA, virus progeny resultingfrom DNA transfections can be better characterized and“over-attenuation” of vaccine viruses can be avoided. For example, thenumber of 132-bp repeats which appears to be associated with attenuation(Maotani et al., 1986) can be exactly determined and, if necessary, bereduced or enlarged according to the needs for vaccine production or thesituation in the field (see further herein). The generation of mutantMDV-1 is greatly facilitated. So far, MDV-1 mutants are generated bylaborious and time-consuming homologous recombination and selectionprocedures in eukaryotic cells. These selection procedures—as reportedfor other herpes viruses—often result in mutations of the genome otherthan those desired, especially since in the case of MDV-1, no cell-freevirus can be obtained which makes selection procedures and recovery andpropagation of mutants even more complicated. In contrast, the inventionprovides a method to manipulate a viral genome based on mutagenesis viaa recE, recT and the recB/C-suppressing λ gam gene present on plasmidpGETrec (Narayanan et al., 1999). The advantages of the system are (i)that only 30 to 50 bp homology arms are needed to target a specificsequence to be deleted, i.e., deletion of any open reading frame can beachieved without the need to clone recombination cassettes, (ii) themethod is very fast, and (iii) the pGETrec vector conferring themutagenesis system and expressing ampicillin resistance is rapidly lostfrom bacterial cells in the absence of ampicillin.

By using the powerful techniques using the so-called E/T cloningprocedures, one-step mutation and selection in Escherichia coli ispossible (Muyrers et al., 1999; Narayanan et al., 1999; Zhang et al.,1998). This technique also allows the deletion of essential MDV-1 geneswithout the need of using complementing cell lines since replication ofmutated MDV-1 genomes as provided herein does not require thetranscomplementation of the essential gene deleted. In addition, cloningprocedures are completely unnecessary.

In another embodiment, the invention provides a method of generatingMDV-1 or other (essentially cell-associated herpes) virus BACs,comprising transforming, for example, Escherichia coli DH10B cells withplasmid pBADαβγ, pGETrec or any other plasmid that inducibly or stablyexpresses recE, recT and the λ gam gene, followed by preparing circularviral DNA from lytically or latently infected cells taken, for example,ex vivo or from cell cultures. In a parallel or separate procedure,linear DNA harboring BAC vector sequences and sequences that allowhomologous recombination of the BAC vector sequences with the viral DNAare provided. This linear DNA can, e.g., be produced by PCR or bylinearizing plasmid DNA. Then, expression of recE, recT and the gam genein the Escherichia coli is provided and electrocompetent cells areprovided (e.g., Sambrook et al., 1989). Viral DNA is then electroporatedtogether with linear DNA harboring the BAC vector sequences intocompetent Escherichia coli. Plating on agar containing appropriateantibiotics provides for the to-be-harvested colony or colonies and BACDNA can be prepared as, for example, described in the detaileddescription herein. Infectivity of cloned viral BAC DNA is checked bytransfection of susceptible cells. Herewith, the invention provides amethod to genetically recombine an essentially host cell-associatedherpes viral genome derived from a host cell or tissue without the needto perform (homologous) recombination in eukaryotic cells, allowing oneto obtain an (if required near complete or complete) infectious genomeor herpes viral nucleic acid derived from field isolates or attenuatedisolates alike.

The method as provided herein will also allow one to further attenuatecandidate vaccine MDV-1 or to generate MDV-1 mutants harboring genes ofother important chicken pathogens. In addition, emerging field MDV-1isolates with possibly different and changing antigenetic properties canbe countered by providing a vaccine based on exchange of the respectivemutated genes between the cloned MDV-1 and the current field isolate(s).These changes—as described above—can be performed with the same E/Tcloning technique and, as such, provide the possibility to react tochanges of MDV-1 in the field very rapidly. An attractive advantage of arecovery of infectious MDV-1 as described herein, however, is the use ofa genome as provided herein as DNA vaccine. Up to now, Marek's diseaseis controlled by application of infected-cell preparations.

These preparations not only contain living cells suspended inDMSO-containing media and the whole variety of cellular antigens, butthey also have to be stored in liquid nitrogen. Consequently, the coldchain has to be maintained from vaccine production to the vaccine userand until administration. In addition, once thawed, the vaccine has tobe administered within a very short period of time and every bird has tobe injected. With MDV-1 genome DNA as provide herein, purification ofthe “vaccine” (DNA) is easily feasible and reproducible. DNA isextremely stable, the maintenance of the cold chain is not necessary,and infectious DNA can be administered by several routes(intramuscularly, intradermally, in-ovo, orally, by the respiratoryroute, and so on) and in different formulations (with and without acarrier, etc.). In addition, the presence of maternal antibodies doesnot interfere with primary injection of the immunogen.

As such, MDV-1 genomes as herein provided allow for the first time thepossibility to produce and engineer highly efficient and biologicallysafe vaccines against a tumorigenic and economically important disease.The invention thus in general provides a method for limiting the risksof an individual on acquiring or fully manifesting a disease caused byan infection with an essentially host cell-associated herpes viruscomprising administering to the individual a vaccine according to theinvention or a genome according to the invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 Schematic illustration of the cloning procedure to generate BACsharboring complete MDV-1 genomes. Shown is the organization of theapproximately 180 kbp MDV-1 genome (A) and the BamHI-restriction map (B)according to Fukuchi et al. (11). The unique short region (Us) and theORFs located in the Us are shown (C and D). A 2.1 and a 3.1 kbp fragmentbordering the Us2 gene (grey boxes) were amplified by PCR and clonedinto plasmid pTZ18R to give rise to recombinant plasmid pDS. The 7.2 kbpBAC vector released from recombinant plasmid pHA1 (15) was inserted intopDS, resulting in plasmid pDS-pHA1 (E). Restriction enzyme sitesaccording to (2) are abbreviated: B=BamHI, E=EcoRI, P=PstI, Pa=PacI,S=SalI.

FIG. 2 Digitally scanned image of an ethidium bromide stained 0.8%agarose gel. DNA isolated from E. coli DH10B clones BAC19, BAC20 andBAC24 was cleaved with BamHI or EcoRI and separated. The restrictionenzyme digests are flanked by the 1 kb ladder (Gibco-BRL). Asterisksindicate additional bands or size variations of individual fragmentsbetween the three BAC clones.

FIG. 3 Digitally scanned image of DNA of 584Ap80C (V), BAC19, BAC20 andBAC24 cleaved with BamHI or EcoRI, separated by 0.8% agarose gelelectrophoresis and stained with ethidium bromide (left panel). AfterSouthern transfer of DNA fragments to Nylon membranes, hybridizationwith Digoxigenin-labeled fragments released from plasmid pDS or pHA1 wasperformed. Size markers (1 kb ladder, Gibco-BRL) and sizes of reactivebands are given.

FIG. 4 Digitally scanned images of Southern blots to analyze sizevariations in BAC19, BAC20 and BAC24 DNA. Viral DNA of strain 584Ap80C(V) and individual BACs was cleaved with BamHI or EcoRI and transferredto Nylon membranes. Sheets were incubated with Digoxigenin-labeled BAC19DNA or labeled BamHI-C or BamHI-D fragments. Size markers (1 kb ladder,Gibco-BRL) are given. The smear-like appearance of bands in the case of584Ap80C DNA when hybridized with BamHI-D sequences is indicated bybrackets.

FIG. 5 (A) IIF analysis of MDV-1 plaques after transfection of BAC19,BAC20 or BAC24 DNA. At 5 days after transfection, infected cells werefixed and subjected to indirect immunofluorescence using anti-gB mab2K11. Detection of bound antibodies was performed with anti-mouse ALEXA™488 dye conjugates (Molecular Probes) and nuclei were counterstainedwith propidium iodide. Magnification=400×.

(B) Growth curves of MDV-1 strain 584A and various BACs. After infectionof CEF cell with 100 p.f.u. of 584Ap80C or transfection progeny ofBAC19, BAC20 or BAC24, virus titers were determined at the indicatedtimes p.i. by co-seeding with fresh CEF cells. Plaques were countedafter immunofluorescent staining with mab 2K11.

FIG. 6 Digitally scanned images of Southern blots to analyze thestability of BAC vetor sequences in viruses recovered after transfectionof BAC19 and BAC20. Transactions progeny was passaged for four times andafter each passage, viral DNA was isolated. Virus DNA was cleaved withBamHI or EcoRI, separated by 0.8% agarose gel electrophoresis andtransferred to Nylon membranes. Southern blot hybridization wasperformed using Digoxigenin-labeled fragments of plasmids pDS or pHA1.Abbreviations: V=584Ap80C, 19=BAC19, 20=BAC20. Passages 1 to 4 aftertransfection of BAC19 DNA are indicated by numbers 1 to 4. Passage 4after transfection of BAC20 DNA was loaded in the last lane,respectively, and is indicated by 4a. Sizes of reactive fragments aregiven. Asterisks indicate the reactive 1.6 kb band of the marker (1 kbladder, Gibco-BRL).

FIG. 7 (A) Schematic illustration of mutagenesis of BAC20 to removegB-encoding sequences. Recombinant plasmid pGETrec encodingL-arabinose-inducible recE, recT, and gam gene was transformed intoBAC20-containing DH10B cells. After PCR amplification of the kan^(R)gene from plasmid pEGFP-N1 (Clontech) with primers that also contained50 nt homology arms bordering the gB deletion, a 1.6 kbp PCR ampliconwas electroporated into DH10B cells harboring BAC20 and pGETrec.Bacterial suspensions were plated on agar containing 30 μg/ml kanamycinand 30 μg/ml chloramphenicol. Double-resistant colonies were picked andsubjected to further analysis.

(B) Schematic illustration of the location of the gB gene in MDV-1 andthe deletion present in BAC 20DgB.

FIG. 8 Scanned image of an ethidium bromide stained 0.8% agarose gelcontaining BAC20 and 20DgB DNA cleaved with BamHI, EcoRI, BgII, or StuIand separated by 0.8% agarose gel electrophoresis (left panel). DNAfragments were transferred to nylon membranes and hybridized with aDigoxigenin-labeled kan^(R−) gB-specific probe. Sizes of reactive DNAfragments are indicated. Abbreviations: B=BamHI, E=EcoRI, Bg=BglI,S=StuI.

FIG. 9 Confocal laser scan analysis of MgB1 cells constitutivelyexpressing MDV-1 gB. MgB1 or QM7 cells were seeded on glass cover slipsand incubated with anti-gB mab 2K11 or convalescent chicken serum MDSI.Secondary antibodies were anti-mouse or anti-chicken IgG ALEXA™ 488 dyeconjugates (Molecular Probes). Nuclei were counterstained with propidiumiodide. Bar represents 10 μm.

FIG. 10 IIF analysis of MgB1, QM7 or CEF cells after transfection withBAC20 (upper panels) or 20DgB (lower panels). At 5 days aftertransfection, cells were fixed with acetone and incubated with anti-pp38mab H19. The secondary antibody was anti-mouse IgG ALEXA™ 488 dyeconjugates. Whereas MDV-1 plaques were observed on all cell lines aftertransfection of BAC20 DNA, viral plaques were only observed on MgB1cells after transfection with 20DgB. Only single infected cells wereobserved on QM7 and CEF cells (arrowheads). Magnification=400 ×.

DETAILED DESCRIPTION OF THE INVENTION

Marek's disease virus (MDV) is a member of the Alphaherpesvirinaesubfamily of the Herpesviridae (van Regenmortel et al., 1999). Based onvirulence for chickens, the ability to induce T cell lymphomas andantigenic properties, MDV is grouped into three serotypes (MDV-1, MDV-2,and MDV-3) (Payne, 1985). MDV-3 represents the herpes virus of turkeys(HVT) which has been widely used for vaccination against MDV-relateddisease. According to the most recent nomenclature, MDV-1 is classifiedas gallid herpes virus 2 (GHV-2), MDV-2 as GHV-3, and HVT as meleagridherpes virus. All three viruses belong to the new Marek's disease-likevirus genus within the Alphaherpesvirinae.

Control of MDV-1 infection was achieved by vaccination primarily withHVT; however, after vaccination failures and description of so-called“very virulent” MDV-1 (Witter, 1989), MDV-2 strains and later attenuatedMDV-1 strains (e.g., strain CVI 988 Rispens) have been used in vaccineformulations (Witter, 1985).

In recent years and first reported in the United States, even morevirulent MDV-1, “very virulent plus” (vv+), and MDV-1 variants appearedand caused high mortality even in vaccinated flocks (Witter, 1997). Oneof these vv+ strains, 584A, was passaged serially on chicken embryofibroblasts (CEF) and lost pathogenicity for chickens (Witter, 1997).The molecular basis for the increased pathogenicity of vv+ MDV-1 andsimilarly for loss of virulence are poorly understood because molecularanalyses of MDV-1 are difficult to perform.

On the one hand, no infectious virus progeny is released in culturedcells; on the other hand, production of MDV-1 recombinants is laboriousand, due to the highly cell-associated nature of the agent in vitro,multiple rounds of purification of virus recombinants are needed(Cantello et al., 1991; Sakaguchi et al., 1993; Parcells et al., 1994,1995; Schat et al., 1998; Anderson et al., 1998). In addition, primarycells have to be used for growth of MDV-1 (Payne), resulting in the factthat analysis of essential MDV-1 genes is almost impossible because notranscomplementing cell lines can be generated.

The genomes of murine and human cytomegaloviruses (MCMV and HCMV;Messerle et al., 1997; Borst et al., 1999), herpes simplex virus type 1(HSV-1; Suter et al., 1999), pseudorabies virus (PrV; Smith et al.,1999, 2000), and Epstein-Barr virus (EBV; Delecluse et al., 1998) havebeen cloned as infectious BACs using this technique.

The aim of this study was to provide a basis for fast and efficientproduction of MDV-1 recombinants by cloning of the complete 180 kbpgenome in Escherichia coli. Infectious MDV-1 was readily recovered aftertransfection of cloned MDV-1 BAC DNA using CEF cells and MDV-1 BACs werestable after several rounds of bacterial growth or serial propagation inCEF cells.

Lastly, because one-step deletion of an essential MDV-1 gene inEscherichia coli was possible, the system may have great potential tofacilitate future analysis of essential and nonessential MDV-1 genes andserve as a tool for production of biologically safe modified live virusand/or DNA vaccines.

The invention is further explained with the aid of the followingillustrative examples.

EXAMPLES Materials and Methods

Virus and cells. Primary or secondary chicken embryo fibroblasts (CEF)or quail muscle (QM7) cells were maintained in Dulbecco's modifiedessential medium (DMEM) supplemented with 5 to 10% fetal calf serum(FCS). MDV-1 strain 584Ap80C was kindly provided by Dr. Richard Witter,ADOL, East Lansing, Mich., U.S.A. Strain 584Ap80C represents anavirulent cell culture passaged descendant of vv+ strain 584A (Witter,1997) and was grown on primary or secondary CEF cells as previouslydescribed (Osterrieder, 1999). QM7 cells were tested for the absence ofMDV-1 sequences by PCR and Southern blot hybridization targetingdifferent regions of the genome before they were used for propagation ofMDV-1 (Tischer, 2002). Virus growth curves were done as described withslight modifications (Parcells et al., 1994). Briefly, 100plaque-forming units (p.f.u.) were used to infect 2×10⁶ freshly seededCEF cells. At various times after infection (0, 12, 24, 48, 72, 96, 120hr), infected cells were trypsinized and titrated on fresh CEF cells.Numbers of plaques were determined and results represent means of twoindependent experiments.

A QM7 cell line constitutively expressing MDV-1 gB was obtained bytransfection of 1×10⁶ QM7 cells with 10 μg of pcMgB (FIG. 1), which isbased on pcDNA3 (Invitrogen) and contains the MDV-1 gB gene from strainRispens CV1988 under the control of the human cytomegalovirus immediateearly promoter. pcMgB-containing QM7 cells were selected in the presenceof 1 mg/ml G418, and gB-expressing clones were identified using anti-gBmonoclonal antibody (mab) 2K11 (kindly provided by Dr. Jean-FrancoisVautherot, INRA, Tours, France). The resulting cell line expressingMDV-1 gB was termed MgB1.

Construction of MDV-1 BACs. MDV-1 DNA was purified from infected cellsby sodium dodecyl sulfate-Proteinase K extraction as described earlier(Morgan et al., 1990). Plasmid pDS-pHA1 was constructed as follows. 2.1and 3.1 kbp fragments on either side of the MDV-1 Us2 gene (FIG. 1) wereamplified by polymerase chain reaction (PCR) using standard primerscontaining appropriate restriction enzyme sites (Table 1), and bothfragments were cloned into pTZ18R (Pharmacia-Amersham). The BAC vectorcontaining the Eco-gpt gene under the control of the HCMV immediateearly promoter was released from plasmid pHA1 (kindly provided by Dr. M.Messerle, LMU Munich, Germany; Messerle et al., 1997) and inserted intothe PacI sites introduced into both the 2.1 and 3.1 kbp fragment presentin plasmid pDS (FIG. 1).

Primary CEF cells were co-transfected with 2 μg 584Ap80C DNA and 10 μgof pDS-pHA1. At 5 days after transfection, cells were plated on primaryCEF cells in the presence of 250 μg/ml mycophenolic acid (MPA), 50 μg/mlxanthine and 100 μg/ml hypoxanthine. The MPA/xanthine/hypoxanthineselection was repeated for a total of four times. After completecytopathic effect (cpe) had developed after the fourth round ofselection, viral DNA was prepared from infected cells and 1 μg ofinfected-cell DNA was electroporated into DHB10 Escherichia coli cells.Colonies were detected from 16 hr after transfection on agar platescontaining 30 μg/ml chloramphenicol (Sambrook et al., 1989). Singlecolonies were picked and BAC DNA was prepared from Escherichia colifollowing a standard alkaline lysis protocol (Sambrook et al., 1989).Large-scale preparation of BAC DNA was performed by silica-basedaffinity chromatography using commercially available kits (Qiagen,Macherey & Nagel). Three MDV-1 584Ap80C BAC clones (BAC19, BAC20, BAC24)were chosen for further analysis.

Mutagenesis of MDV-1 BACs. For mutagenesis of cloned MDV-1 DNA inEscherichia coli, recE-catalyzed reactions promoting homologousrecombination between linear DNA fragments, referred to as E/T cloning,was performed (Zhang et al., 1998; Narayanan et al., 1999). PlasmidpGETrec (kindly provided by Dr. Panos Ioannou, Murdoch Institute,Melbourne, Australia) harboring the recE, recT and bacteriophage 1 gamgene was transformed into BAC20-containing DH10B cells (Narayanan etal., 1999). After induction of recE, recT and gam by addition of 0.2%arabinose, electrocompetent cells were prepared essentially as described(Narayanan). To delete the gB gene in BAC20, the kanamycin resistancegene (kan^(R)) of plasmid pEGFP-N1 (Clontech) was amplified by PCR. Thedesigned primers contained 50 nucleotide homology arms bordering thedesired deletion within gB and 20 nucleotides for amplification ofkan^(R) (Table 1). The resulting 1.6 kbp fragment was purified from anagarose gel (Qiagen) and electroporated in pGETrec-containing BAC20cells. Colonies harboring the cam^(R) and kan^(R) genes were identifiedon plates containing both antibiotics (Narayanan et al., 1999).

DNA analyses. BAC or viral 584Ap80C DNA was cleaved with EcoRI, BamHI,BglII or StuI and separated on 0.8% agarose gels. DNA fragments weretransferred to positively charged Nylon membranes (Pharmacia-Amersham)and Southern blot hybridization was performed using Digoxigenin-labeledBAC19 DNA or individual BamHI fragments of MDV-1 strain GA(Katsuragi-Iwanaga, 1990; Osterrieder, 1999).

In addition, a gB-specific probe from plasmid pcgB and a probe harboringthe kan^(R) gene were prepared for analysis of gB-negative MDV-1 BAC.Chemoluminescent detection of DNA hybrids using CSPD™ was done accordingto the manufacturer's instruction (Roche Biochemicals).

Indirect immunofluorescence. For indirect immunofluorescence analyses(IIF), cells were grown on 6- or 24-well plates (Greiner Bio-One) or onglass cover slips and subsequently infected where indicated. Cells werefixed with 90% acetone at various times after infection or transfection,and IIF was done exactly as described (Meindl and Osterrieder, 1999).Samples were analyzed by fluorescence microscopy or confocal laserscanning microscopy (CLSM). The antibodies used were anti-gB mab 2K11,anti-pp38 mab H19 (kindly provided by Dr. Lucy Lee, ADOL, East Lansing,Mich.) or a convalescent serum from a chicken infected with MDV-1(MDSI).

Results:

Construction and analysis of BACs containing complete MDV-1 genomes. Onemillion primary CEF were infected with 1×10⁴ p.f.u. of MDV-1 strain,i.e., infected cells were mixed with uninfected cells. After completecytopathic effect had developed, DNA was prepared from infected cellsand 2 μg viral DNA were transfected into 1×10⁶ primary CEF cellstogether with 10 μg of pDS-pHA1 plasmid DNA. Five days aftertransfection, cells were co-seeded with fresh CEF and overlaid withselection medium.

This procedure was repeated for a total of four times. Finally, DNA fromrecombinant MDV-1 that was able to grow in the presence ofMPA/xanthine/hypoxanthine was isolated and subjected to Southern blotanalysis using labeled pHA1 as a probe. It could be demonstrated that aportion of the viral DNA contained inserted F plasmid sequences (datanot shown). One microgram of this viral DNA was used to transformEscherichia coil DH10B cells. Transformed bacteria were plated on agarcontaining 30 μg/ml chloramphenicol and single colonies were picked. DNAof bacterial colonies was extracted by standard plasmid preparationprocedures (Sambrook et al., 1989) and run on 0.8% agarose gels.

Several of the bacterial colonies were shown to contain high molecularweight extrachromosomal DNA, and three of the clones (BAC19, BAC20 andBAC24) were chosen for further analysis (FIG. 2). To furthercharacterize the isolated BAC clones, Southern blot analysis of 584Ap80Cand BAC DNA after cleavage with BamHI or EcoRI with labeled BAC19 DNA asa probe was performed. It could be demonstrated that BAC19, BAC20, andBAC24 DNA exhibited almost identical restriction enzyme fragmentpatterns when compared to that of the parental 584Ap80C (FIG. 3). Twonotable exceptions, however, were readily recognized. The 20 kbp BamHI-Afragment present in 584Ap80C DNA was absent in all analyzed BAC clones.Instead, fragments of 16 and 10 kbp in size were detected in DNA ofBAC19, BAC20 and BAC24 (FIG. 3). These two bands represented theenlarged BamHI-A fragment in which, by virtue of insertion of the Fplasmid and deletion of Us2 sequences, an additional BamHI site wasintroduced (FIG. 1).

In EcoRI-digested BAC DNA, one additional band of 5.8 kbp (BACsequences) and minor alterations in sizes of fragments caused by thedeletion of the Us2 gene were observed (FIGS. 1 and 3). The correctinsertion of the BAC sequences in the various clones was furtheranalyzed by Southern blot hybridizations using labeled inserts ofplasmid pDS or pHA1 as a probe, and the expected reaction pattern inBamHI- or EcoRI-digested DNA was observed. In BamHI-digested BAC DNAs,16 and 10 kbp BamHI fragments specifically reacted with the pDS probewhereas only the 10 kbp fragment was reactive with a probe derived fromplasmid pHA1 (FIG. 1; FIG. 3).

In EcoRI-digested BAC19, BAC20 or BAC24 DNA, fragments of 4.3, 2.8 and1.7 kbp specifically reacted with the pDS probe, whereas 5.8 and 1.7 kbpfragments specifically hybridized with the pHA1 probe (FIG. 1, FIG. 3).These fragments exactly corresponded to those predicted after insertionof the pHA1 sequences (FIG. 1), and it was concluded that the F plasmidsequences were correctly inserted instead of the Us2 ORF in all MDV-1BACs analyzed. In addition, some variation in banding patterns of BAC19,BAC20, and BAC24 was noted in either BamHI- or EcoRI-digested DNA, e.g.,an additional band of approximately 6.2 kbp in BamHI-digested BAC19 DNAor additional bands in EcoRI-digested DNA of BAC20 and BAC24 (FIGS. 2,3). To address the question of the observed size variations ofindividual restriction enzyme fragments, hybridization with the labeledBamHI-D fragment was performed because size variations in the terminaland internal repeats of the unique long region (TRL and IRL) are common.

It was shown by Southern blotting that the additional fragments observedin either BamHI- or EcoRI-digested DNA of BAC19, BAC20, or BAC24resulted indeed from variations in TRL and IRL. Whereas two broad smearswith the BamHI-D probe were detected in viral 584Ap80C DNA digested withBamHI which ranged from approximately 9 to 15 kbp and from 4 to 8 kbp(corresponding to the BamHI-D and -H fragments of virulent MDV-1,respectively; FIG. 1), distinct but different bands were observed in allBAC clones analyzed (FIG. 4). All other restriction enzyme fragments ofthe different BAC clones appeared to be identical with those of viral584Ap80C DNA. This was confirmed by using several other labeled BamHIfragments as probes, including BamHI-A, -B, -C and -I₂ fragments (datafor the BamHI-C probe are exemplarily shown in FIG. 4).

Reconstitution of infectious MDV-1 from cloned DNA. DNA of BAC19, BAC20or BAC24 was transfected into primary CEF. At 3 to 7 days aftertransfection, MDV-1 specific virus plaques appeared as demonstrated byIIF using anti-MDV-1 gB mab. MDV-1 rescued after transfection of thevarious BACs was then co-seeded with fresh CEF and sizes of plaques werecompared to those induced by parental 584Ap80C. As exemplarily shown forplaques stained on day 2 p.i., no appreciable differences in plaquesizes between recombinant and parental viruses were detected (FIG. 5A).

To further characterize the biological properties of MDV-1 recoveredafter BAC transfection, growth kinetics of these viruses were comparedto that of parental 584Ap80C. In the case of BACs, virus recovered atday 5 after transfection were used to infect fresh CEF cells seeded on6-well plates (50 p.f.u. of virus were used to infect one wellcontaining 1×10⁶ cells). Similarly, 50 p.f.u. of 584Ap80C were used toinfect fresh CEF in the same way. At various times p.i., virus washarvested and titrated by co-seeding 10-fold virus dilutions with freshCEF cells. The results of these experiments are summarized in FIG. 5B.

It could be demonstrated that all MDV-1 BACs tested exhibited growthcharacteristics that were almost identical to those of parental 584Ap80C(FIG. 5B). Maximal titers were reached at 72 hr p.i. and remainedvirtually constant until the end of the observation period at 120 hrp.i. From the plaque sizes and growth characteristics we concluded thatthe biological properties of MDV-1 BACs in vitro were virtuallyindistinguishable from those of the parental strain.

To ascertain stability of the BAC-derived viruses, progeny of BACtransfections of BAC19 and BAC 20 was passaged four times and viral DNAwas prepared. Viral DNA was cleaved with BamHI or EcoRI, separated by0.8% agarose gel electrophoresis, and transferred to Nylon membranes.Hybridization was performed using the pDS or the pHA1 probe. Similar DNAfragments as described above were observed and the banding pattern didnot change with serial passage of transfection progeny as analyzed withthe two probes (FIG. 6). From these results we concluded that Fplasmid-derived sequences remained stably inserted within the 584Ap80Cgenomes recovered from individual MDV-1 BAC clones even after serialpassage in CEF cells.

However, as shown by hybridization with the BamHI-D fragment and PCRanalysis, variability of the 132-bp repeat sequences was restored and adiffuse smear of reactive bands was observed in BamHI- or EcoRI-cleavedDNA of transfection progeny already after the first virus passage (datanot shown).

Mutagenesis of BAC20 and deletion of gB-encoding sequences. In the nextexperiments, a recently developed method for mutagenesis of BACs wasapplied to remove 2.3 kbp of the 2.8 kbp gB gene from BAC20 (FIG. 7).After transformation of plasmid pGETrec (Narayanan) intoBAC20-containing DH10B, the kan^(R) gene was amplified with primers thatallowed homologous recombination with MDV-1 gB sequences (Table 1; FIG.8) and electroporated into BAC20-pGETrec cells. Bacteria were plated onLB agar containing chloramphenicol and kanamycin, and double resistantcolonies were picked. After DNA isolation of individual colonies,Southern blot analysis of recombinant BAC20 harboring a deletion withinthe gB gene (20DgB) was performed. A kan^(R−) and a gB-specific probedetected fragments of 20DgB after cleavage with BamHI, EcoRI, BglII orStuI that were in perfect agreement with those calculated afterinsertion of the kan^(R) resistance gene into gB-encoding sequences(FIG. 9). It was noted that—as reported previously—pGETrec, whichconfers ampicillin resistance, was easily lost from Escherichia colicells grown in the absence of the antibiotic (FIG. 9). From theseresults we concluded that the gB open reading frame was almostcompletely removed from 20DgB.

Analysis of gB-negative MDV-1 reconstituted from 20DgB. Because gB isessential for growth of all herpes viruses analyzed to date (reviewed inPereira), a QM7 cell line which expressed MDV-1 gB under the control ofthe HCMV immediate early promoter was generated. Indirectimmunofluorescence analyses demonstrated that virtually every cell ofcell line MgB1 constitutively expressed MDV-1 gB as demonstrated usingmab 2K11 or a convalescent chicken serum (MDSI) (FIG. 10). To analyzegrowth of BAC20 and 20DgB in various cell lines, DNA was prepared andused to transfect CEF, QM7 or MgB1 cells. At 3 to 5 days aftertransfection, virus plaques were observed in all cells transfected withBAC20 (FIG. 10).

However, after transfection of 20DgB DNA, plaques were observed ingB-expressing MgB1 cells only (FIG. 10). In CEF and QM7 cellstransfected with 20DgB, single cells expressed the early pp38 gene asdemonstrated by reactivity with mab H19 (Lee et al., but plaqueformation was inhibited (FIG. 10). These results of gB being essentialfor MDV-1 cell-to-cell spread in vitro were confirmed by co-seeding20DgB-infected MgB1 cells with CEF, QM7 or fresh MgB1 cells. As shownafter primary transfection, plaque formation was only observed afterco-seeding with gB-expressing cells. From these results we concludedthat MDV-1 gB is essentially required for cell-to-cell spread of MDV-1in cultured cells.

Although Marek's disease virus is an important pathogen of chickens thatcauses T cell tumors and high mortality in infected animals, little isknown about the function of individual genes and gene products in thelytic, latent or tumor phase of the infection. Analysis of MDV-1 genesand gene products has been greatly impaired for two main reasons.Firstly, cultured cells infected with MDV-1 do not yield free infectiousvirus, and secondly, efficient growth of MDV-1 in cultured cells isrestricted to primary or secondary chicken embryo fibroblasts.

Hence, mutagenesis using conventional homologous recombination used tomutagenize other Alphaherpesvirinae is laborious, time-consuming andrequires constant supply of primary cells. While mutagenesis of HSV andPrV is certainly facilitated by using the BAC technology, conventionalmutagenesis relying on homologous recombination in eukaryotic cellsrepresents a standard technique for these two viruses and numerousmutant viruses have been generated. In contrast, for mutagenesis ofMDV-1, BAC cloning and mutagenesis is a major advantage. Once the MDV-1genome is cloned as a BAC and can be stably maintained in Escherichiacoli, generation of mutants and analyses of essential genes should berelatively easy. In fact, it was possible to clone the complete genomeof strain 584Ap80C as an infectious BAC. Strain 584Ap80C is a descendantof the very virulent plus (vv+) MDV-1 strain 584A after 80 serialpassages on CEF cells (Witter, 1997). Analysis of the cloned MDV-1genomes present in BAC19, BAC20 and BAC24 demonstrated that variationsof restriction enzyme patterns were obvious.

This heterogeneity could be attributed to variations in the BamHI-D and-H fragments. It is known that varying numbers of 132-bp tandem repeatsare present in various MDV-1 strains and that the number of repeatsincreases after serial passage in cultured cells (Maotani, Silva,Fukuchi). In addition, the number of the tandem 132-bp repeats wasassociated with a loss of oncogenicity because a constant number ofthese units was demonstrated in virulent strains (Fukuchi et al., 1985;Bradley et al., 1989), although recent work on the widely used RispensCVI 988 vaccine strain demonstrated that there might be no directcorrelation of small numbers of the 132-bp repeats and virulence. In thecase of MDV-1 strain 584Ap80C, hybridization of restrictionenzyme-digested viral DNA with the BamHI-D fragment yielded diffusebanding patterns indicating a variable number of repeats present in thevirus population. In contrast, only single strongly reactive bands wereidentified in each of the BAC clones with the same probe. However, thesizes of reactive bands after cleavage with BamHI or EcoRI variedbetween BAC19, BAC20 and BAC24, indicating that genomes containingdifferent numbers of the 132-bp repeats had been cloned. Thisinterpretation was substantiated by PCR analyses targeting the 132-bprepeats. Whereas the typical ladder-like appearance of PCR products wasobtained with DNA of 584Ap80C (Becker et al., 1993), distinct bands wereamplified from cloned viral DNA in the case of BAC19, BAC20 or BAC24.

It was therefore concluded that the variable restriction enzyme patternsof the different BAC clones resulted from various numbers of tandem132-bp repeats present in the individual clones which did not influencethe infectivity of the cloned DNA because infectious virus was recoveredafter transfection of DNA isolated from each of the different BACclones.

After cloning of the complete MDV-1 genome and proof of infectivity ofcloned MDV-1 DNA, a recently developed mutagenesis system in which alinear DNA fragment can be recombined into bacteria-resident DNA andwhich is catalyzed by recE (Narayanan, Muyrers) was used to deletegB-encoding sequences of BAC20. The mutagenesis is based on recE, recTand the recB/C-suppressing λ gam gene present on plasmid pGETrec(Narayanan et al., 1999).

The big advantages of the system that was used for the first time tomanipulate a virus genome are: (i) only 30 to 50 bp homology arms areneeded to target a specific sequence to be deleted, i.e., deletion ofany open reading frame can be achieved without the need to clonerecombination cassettes, (ii) the method is very fast, and (iii) thepGETrec vector conferring the mutagenesis system and expressingampicillin resistance is rapidly lost from bacterial cells in theabsence of ampicillin. After electroporation of the gB knockout PCRproduct into pGETrec-containing BAC20 cells, between 10 and 30 cam^(R)and kan^(R) double-resistant colonies were obtained. One of the colonieswas termed 20DgB-1 and chosen for further analyses because it had lostpGETrec immediately after plating on agar containing chloramphenicol andkanamycin.

Southern blot analyses demonstrated successful deletion of the gB geneand the insertion of the kan^(R) gene in 20DgB-1. MDV-1 recovered aftertransfection of CEF cells with 20DgB-1 was unable to spread frominfected cells to neighboring cells, indicating that MDV-1 gB, like itscounterparts in other herpes viruses, is essential for cell-to-cellspread of infectivity. Because MDV-1 is highly cell-associated incultured cells and does not release infectious virus to the culturemedium, we were not able to investigate a possible role of MDV-1 gB invirus entry. The generated gB mutant represents the first example of anMDV-1 with deletion of an essential gene and demonstrates the power ofthe BAC cloning and mutagenesis system which is especially useful in thecase of MDV-1. Using MDV-1 BAC and the permanent cell line QM7 whichallows MDV-1 propagation and which—unlike quail fibroblast QT35 cellline—does not harbor MDV-1 sequences (Majerciak, 2001), represents anexcellent combination to thoroughly analyze essential MDV-1 genes. Inaddition, comparative analyses on gene functions of variousAlphaherpesvirinae can now include MDV-1 and allows studies on verydistantly related members, such as VZV or BHV-4 of one viral family.

With the cloned genomes as provided herein in at hand, a detailedfurther assessment of lytic, latent and tumor-inducing genes is providedfor virus in which genetic manipulations used to be very restricted.

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TABLE 1 Primers used for generation of plasmids pDS and pcMgB and fordeletion of gB Fragment/plasmid Primer Sequence Generated MUS215′-ACAggattcCGTGTTTGAATACTGG-3′^(a) (SEQ ID NO:1) 2.1 kb pDS MUS225′-ATAgtcgacTttaattaaCCGGTAGTCATTAGC-3′ (SEQ ID NO:2) 2.1 kb pDS MUS235′-ATCgcatgcTTAATTAATTTGGCAAAACGGAATAGG-3′ (SEQ ID NO:3) 3.1 kb pDSMUS24 5′-CGCaagcttAATATGAATCTCTAAAACTTCTCGGC-3′ (SEQ ID NO:4) 3.1 kb pDSgB-up 5′-GATAgaatccATGCACTATTTTAGGCGG-3′ (SEQ ID NO:5) pcMgB gB-low5′-ATACctcgagTTACACAGCATCATCTTCTG-3′ (SEQ ID NO:6) pcMgB gBkana 5′-

(SEQ ID NO:7) kan^(R) gene for gB

AAAGCACTAAATCGGAACCCTAAAGGGAGC-3′^(b) deletion gBkanb 5′-

(SEQ ID NO:8) kan^(R) gene for gB

ATTGTCTCCTTCCGTGTTTCAGTTAGCCTC-3′ deletion ^(a)Bold sequences indicaterestriction enzyme sites, sequences in italics indicate additional basesnot present in the MDV-1 sequence. ^(b)Underlined sequences indicatesequences from pEGFP-N1 used to amplify the kan^(r) gene, bold andunderlined sequences indicate gB sequences used for homologousrecombination and recE/T-mediated deletion of gB.

What is claimed is:
 1. A composition comprising: a genomic nucleic acidmolecule comprising a replicative full-length copy of a Marek's DiseaseVirus-1 (MDV-1) herpesvirus genome, except in that the US2 codingsequence of the MDV-1 herpesvirus genome is replaced by a bacterialartificial chromosome (BAC) vector; wherein said genomic nucleic acidmolecule is capable of replication in a eukaryotic host cell; whereinsaid genomic nucleic acid molecule encodes infectious MDV-1 herpesviruswhen the genomic nucleic acid molecule is expressed in a host cell; andwherein said BAC vector allows for recombination of the genomic nucleicacid in a bacterium.
 2. The composition of claim 1 wherein said MDV-1herpesvirus genome has an additional functional deletion in a genedifferent from US2.
 3. The composition of claim 1 wherein saidreplicative genome is of a MDV-1 herpesvirus that is a virulent, a veryvirulent, or a very virulent plus field virus.
 4. A method of inducingan immune response in a subject, said method comprising: administeringto the subject a composition of claim
 1. 5. A method for limiting therisks of a subject acquiring or fully manifesting a disease caused by aninfection with a strictly host cell-associated Marek's Disease Virus-1(MDV-1) herpesvirus, said method comprising administering to the subjectthe composition of claim
 1. 6. The method according to claim 5 whereinsaid subject is a bird.
 7. A nucleic acid comprising: a nucleotidesequence that is a replicative genome of a Marek's Disease Virus-1(MDV-1) herpesvirus; and a bacterial artificial chromosome (BAC) vectorsequence; wherein the replicative genome of a Marek's Disease Virus-1(MDV-1) herpesvirus comprises a full-length copy of a MDV-1 herpesvirusgenome except in that the US2 coding sequence is replaced by the BACvector; wherein the nucleic acid is capable of replication in aeukaryotic host cell; wherein the nucleic acid encodes infectious MDV-1herpesvirus when the nucleic acid is expressed in a host cell; andwherein the BAC vector sequence allows for recombination of the nucleicacid in a bacterium.